PROJECT 4: Skeletal Muscle Loss and Dysfunction ? SUMMARY LeBrasseur In line with the overall goal of the Program Project Grant, Project 4 will test the central hypothesis that senescent cells mechanistically contribute to skeletal muscle aging and represent a novel druggable target to restore muscle performance, physical function, and organismal resilience. Our hypothesis is founded on our recent work demonstrating the role of cellular senescence, a hallmark of aging, in the genesis of multiple age- related conditions. Our preliminary data demonstrate expression of the cyclin-dependent kinase inhibitor p21Cip1, a marker and mediator of senescence, increases in aged murine and human skeletal muscle and negatively associates with measures of physical function. We show that p21Cip1, senescence-associated secretory phenotype (SASP), and anti-apoptotic proteins markedly increase in cultured myoblasts in response to senescence-inducing stress. Aged muscle is compositionally heterogenous, however, and senescence of other resident cell populations, including fibroadipogenic progenitor, endothelial, and immune cells, may also contribute to its degeneration. Consequently, there is a critical need to identify and comprehensively phenotype the cell populations within aged muscle that senesce and mechanistically contribute to its loss and dysfunction. To this end, in Aim 1 we will use mice harboring a transgene that enables the isolation of p21Cip1-expressing cells to quantify markers of senescence, the SASP, and anti-apoptosis pathways in muscle-resident cells of young and aged mice. High dimensional mapping of non-senescent and senescent cell populations will be accomplished through mass cytometry and advanced histological approaches. Aim 2, will directly compare the effects of genetic clearance of p21Cip-expressing cells to clearance of p16Ink4a-expressing cells on muscle health (e.g., mass, fibrosis, and fat infiltration) and measures of physical function and resilience with the support of Integrated Healthspan Phenotyping Core. We will also assess the relative efficacy of clearing specific p21Cip1-cell populations using novel Cre-LoxP lines and pharmacological agents developed and screened by the Drug Discovery and Development Core. Finally, Aim 3 will test the hypothesis that genetic and pharmacological clearance of senescent cells will potentiate the effects of a muscle building drug on measures of muscle health, physical function, and resilience. Secondary analyses will include the effects on metabolic, bone, and cardiovascular health in partnership with Projects 1, 2, and 3, respectively. Through the use of novel analytical, transgenic, and pharmacological tools and a multidisciplinary approach, we expect to advance our understanding of the fundamental biology of skeletal muscle aging. The application of clinically-relevant measures of physical function and resilience and evidence-based senotherapeutic compounds will facilitate the translation of preclinical discoveries to clinical application.